[4] Caged fluorescent probes

Mitchison, Tim; Sawin, Kenneth E.; Theriot, Julie A.; Gee, Kyle R.; Mallavarapu, Aneil

doi:10.1016/s0076-6879(98)91007-2

Cited by 137 publications

(119 citation statements)

References 15 publications

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“…CMNCBZ-caged lysine (67,68) Electrophysiology. The stellate ganglion was dissected from Loligo pealei, and recordings of synaptic transmission were done as described (69,70).…”

Section: Methodsmentioning

confidence: 99%

Photolysis of a caged peptide reveals rapid action of N -ethylmaleimide sensitive factor before neurotransmitter release

Kuner

Gee³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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The time at which the N-ethylmaleimide-sensitive factor (NSF) acts during synaptic vesicle (SV) trafficking was identified by timecontrolled perturbation of NSF function with a photoactivatable inhibitory peptide. Photolysis of this caged peptide in the squid giant presynaptic terminal caused an abrupt (0.2 s) slowing of the kinetics of the postsynaptic current (PSC) and a more gradual (2-3 s) reduction in PSC amplitude. Based on the rapid rate of these inhibitory effects relative to the speed of SV recycling, we conclude that NSF functions in reactions that immediately precede neurotransmitter release. Our results indicate the locus of SNARE protein recycling in presynaptic terminals and reveal NSF as a potential target for rapid regulation of transmitter release.caged probes ͉ exocytosis ͉ synaptic transmission ͉ synaptic vesicle cycle N eurotransmitter release relies on the precisely coordinated actions of many proteins that serve to recruit synaptic vesicles (SVs) to active zones, prepare SVs for Ca 2ϩ -dependent exocytosis, and recycle used components (1-5). At the core of these trafficking reactions lies the SNARE [soluble Nethylmaleimide sensitive factor (NSF) attachment protein receptor] complex, which consists of proteins present in SVs (v-SNAREs) and the plasma membrane (t-SNAREs) (6). It is thought that trans-SNARE complexes bridging the SV and plasma membranes bring these two membranes into close apposition and mediate membrane fusion (7,8). Because SNARE complexes are highly stable, hydrolysis of ATP by the molecular chaperone NSF (9, 10) is required to disassemble used SNARE complexes and, thereby, recycle SNARE proteins in preparation for future rounds of exocytosis (11-13). Although it is generally agreed that this action of NSF is important for neurotransmitter release, it is not clear whether NSF works before or after vesicle fusion. This distinction is critical for understanding the dynamic control of synaptic transmission by NSF and elucidating the life cycle of SNARE complexes during SV trafficking.Two models have been proposed for the timing of NSF action during neurotransmitter release (Fig. 1). SNAREs could be disassembled just before fusion, meaning that NSF is active only when needed for a fusion reaction (Fig. 1 A). This model is consistent with observations that NSF is required before vesicle fusion in several experimental systems (14-20). Alternatively, NSF could dissociate SNARE complexes immediately after neurotransmitter release (Fig. 1B). Such a postfusion action of NSF could provide an attractive mechanism for sorting of v-and t-SNAREs after fusion: in this case, newly separated v-SNAREs would be carried along as recycled SVs bud from the plasma membrane, whereas t-SNAREs would remain behind in the plasma membrane. Although experimental evidence supporting this conclusion is limited (21,22), the ability to explain SNARE sorting makes a postfusion action of NSF part of most current models of SV trafficking (8,(23)(24)(25)(26).One way to distinguish between these two alternatives ...

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“…CMNCBZ-caged lysine (67,68) Electrophysiology. The stellate ganglion was dissected from Loligo pealei, and recordings of synaptic transmission were done as described (69,70).…”

Section: Methodsmentioning

confidence: 99%

Photolysis of a caged peptide reveals rapid action of N -ethylmaleimide sensitive factor before neurotransmitter release

Kuner

Gee³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…To facilitate the detection of microtubule ends it is also possible to create artificial systems, such as cytoplasts, by cell enucleation, in order to obtain samples with reduced thickness and microtubule density [Komarova et al, 2002]. Additionally using uncaging or photobleaching methods it is possible to either selectively highlight a subset of microtubules [Mitchison et al, 1998] or to create a dark area in which newly growing labelled microtubules can be easily followed [Komarova et al, 2002].…”

mentioning

confidence: 99%

Automatic quantification of microtubule dynamics enables RNAi‐screening of new mitotic spindle regulators

et al. 2011

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The genetic integrity of every organism depends on the faithful partitioning of its genome between two daughter cells in mitosis. In all eukaryotes, chromosome segregation requires the assembly of the mitotic spindle, a bipolar array of dynamic microtubules. Perturbations in microtubule dynamics affect spindle assembly and maintenance and ultimately result in aberrant cell divisions. To identify new regulators of microtubule dynamics within the hundreds of mitotic hits, reported in RNAi screens performed in C. elegans, Drosophila and mammalian tissue culture cells [Sonnichsen et al., 2005; Goshima et al., 2007; Neumann et al., 2010], we established a fast and quantitative assay to measure microtubule dynamics in living cells. Here we present a fully automated workflow from RNAi transfection, via image acquisition and data processing, to the quantitative characterization of microtubule behaviour. Candidate genes are knocked down by solid‐phase reverse transfection with siRNA oligos in HeLa cells stably expressing EB3‐EGFP, a microtubule plus end marker. Mitotic cells are selected using an automatic classifier [Conrad et al., 2011] and imaged on a spinning disk confocal microscope at high temporal and spatial resolution. The time‐lapse movies are analysed using a multiple particle tracking software, developed in‐house, that automatically detects microtubule plus ends, tracks microtubule growth events over consecutive frames and calculates growth speeds, lengths and lifetimes of the tracked microtubules. The entire assay provides a powerful tool to analyse the effect of essential mitotic genes on microtubule dynamics in living cells and to dissect their contribution in spindle assembly and maintenance.

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“…Moreover, photoswitching requires the presence of thiol reagents at high concentrations, which may not be compatible with the live cell environment. Irreversibly photoactivatable dyes for super-resolution imaging are also available, e.g., Q-rhodamine (Mitchison et al 1998;Gee et al 2001).…”

Section: Fluorophoresmentioning

confidence: 99%